EP0185573B1 - Expression and excretion of polypeptides in eucaryotes under the control of an adenovirus promoter - Google Patents

Abstract

Recombinant DNA modified by a nucleotide sequence coding for a specific polypeptide sequence whose expression is sought, this recombinant DNA being suitable for the transformation of eukaryot, especially human or animal, cell lines whose endogenic polymerases are capable of recognising adenovirus promoters. The DNA according to the invention is more particularly characterised in that the said nucleotide insertion sequence is placed under the direct control of the early promoter of the E1A region of the adenovirus genome.

Description

The invention relates to a recombinant DNA comprising a nucleotide sequence coding for a determined polypeptide under the control of an adenovirus promoter, vectors containing this recombinant DNA and eukaryotic cells transformed by this recombinant DNA, the products of excretion of these transformed cells and their applications, in particular to the constitution of vaccines.

Human adenoviruses have a long, linear, double-stranded genome (approximately 36,000 bp) that codes for at least 30 proteins. The viral cycle, during infection of permissive cells, is divided into two phases, early and late. It is known that the four regions of the viral genome expressed in the early phase are called regions E1, E2, E3 and E4, the respective positions of which in the entire viral genome are schematically represented in FIG. 1. The E1 region, located at the left end of the genome, is itself divided into two regions E1A and E1B. The transition from the early to the late phase, marked by the replication of viral DNA, is characterized by a sudden change in the genetic program of the virus. The expression of certain early genes is repressed whereas the transcription of late genes is mainly carried out from a single promoter, the major late promoter (fig. 1). In addition, there is a strong repression of protein synthesis in the host cell (1).

The genetic organization of human adenoviruses type 2 or 5 (Ad2, Ad5) is sufficiently known that their genome can be manipulated in vitro and its use as a vector for expression of a foreign gene in an animal cell in culture has already been envisaged. It is indeed known that the E3 region, which represents 6% of the genome, is not essential in vitro and can therefore be substituted in its entirety (2). The size of the foreign DNA fragment that can be inserted into the genome of these viruses is large. Indeed, the virus can encapsulate a genome whose length exceeds by 5% that of the wild genome.

The promoter of the early E1A region of an adenovirus, transported in mouse cells in which replication of the retroviruses is not possible, has been shown to be able to exert its promoter activity there nevertheless. Thus a prokaryotic gene coding for chloramphenicol acetyltransferase could it be expressed in such a cellular system under the control of said promoter (Kruczek I. et al (1983), Proc. Natl. Acad. Sci. USA).

Different vectors derived from type 2 or 5 adenoviruses have therefore been constructed. In these recombinants, the foreign gene was expressed under the control of the late major promoter. This made it possible in certain cases to obtain a synthesis of the protein encoded by a foreign gene at a level comparable to that of late viral proteins (4, 5, 6, 7), with low in vivo translation efficiencies, in particular eu with regard to the efficiency of transcription into RNAs also observed (6). Additionally the expression of the foreign gene under control of the late promoter can only manifest itself in the late phase of the viral cycle.

The invention stems from the observation that the promoter of the early E1A region of the genome of an adenovirus (hereinafter simply referred to as "E1A promoter") could particularly effectively control the expression in a viral vector of a gene. heterologous (that is to say foreign with respect to the genes which are normally associated with it in the adenovirus) or more generally of a heterologous nucleotide sequence coding for a polypeptide sequence whose expression is sought. Under the conditions defined below, the E1A promoter behaves like a strong promoter, and this more particularly when the E1A promoter-heterologous coding sequence assembly is inserted into this viral vector.

The invention stems from the observation that the promoter of the early E1A region of the genome of an adenovirus (hereinafter simply referred to as "E1A promoter") could particularly effectively control the expression in a viral vector of a gene. heterologous (that is to say foreign with respect to the genes which are normally associated with it in the adenovirus) or more generally of a heterologous nucleotide sequence coding for a polypeptide sequence whose expression is sought. Under the conditions defined below, the E1A promoter behaves like a strong promoter, and this more particularly when the E1A promoter-heterologous coding sequence assembly is inserted into this viral vector.

The invention therefore generally relates to a recombinant vector for the transformation of lines eukaryotic cells, in particular human or animal, chosen from those which are infectable by adenoviruses or whose endogenous polymerases are capable of recognizing the promoters of adenoviruses, this vector being further modified by an insertion nucleic acid containing a nucleotide sequence coding for a polypeptide sequence the expression of which in said cell lines is sought, characterized in that said insertion sequence is substituted for the E1A genes normally placed under the direct control of said early promoter of the E1A region, this insertion sequence being itself even placed under the direct control of this promoter and in that it comprises, downstream of the nucleic acid of insertion, all of the essential sequences of an adenovirus genome which are necessary for the replication of the corresponding adenoviruses that are normally located downstream of genes normally under the control of direct from the early promoter E1A in said genome, and in that the aforesaid nucleotide sequence coding for the polypeptide sequence whose expression is sought is heterologous with respect to the genes of the said adenovirus.

Being a viral vector, derived from an adenovirus, one can also benefit from the advantage attaching to the E1A region of adenoviruses, namely that its expression is constitutive and permanent throughout the viral cycle ( 8, 9).

A particular preferred form of the recombinant DNA according to the invention is characterized in that it comprises, downstream of the nucleic acid of insertion, in the direction of transcription, a defective adenovirus genome nevertheless comprising l 'all of those essential sequences necessary for the replication of the corresponding adenovirus, which are normally located downstream of the genes normally under the direct control of the early promoter E1A in said genome.

Advantageously, the defective adenovirus genome with which the recombinant DNA in accordance with the invention is associated, consists of a complete genome of adenovirus, however devoid of the anterior part of the E1A region of the viral genome, in particular of its fragment. 0-2.6% (percentage expressed relative to the total size of the adenovirus genome).

The recombinant DNAs of the invention, associated with vector elements such as those which have been mentioned above, in fact constitute vectors containing said recombinant DNAs. As far as they are concerned, reference will also be made to "defective recombinant viruses" when the vector elements associated with the DNA-recombinant according to the invention are derived from a defective adenovirus genome. These defective recombinant viruses are advantageously used for the transformation of transformable cell lines of higher eukaryotes (in particular of human or animal origin) themselves comprising a distinct nucleotide sequence capable of complementing the part of the genome of the adenovirus including the above vector is lacking, said distinct sequence preferably being incorporated into the genome of cells of said cell line.

As a preferred example of such cell lines, mention will be made of line 293, a human embryonic kidney line which contains, integrated into its genome, the first eleven percent of the left end of the genome of an Ad5. These make it possible to complement defective recombinant viruses which carry deletions from this region (10).

The use of these recombinant defective virus vector systems - cells containing a sequence capable of complementing the defective recombinant viruses, is of very particular interest, when the nucleotide sequence contained in the nucleic acid of insertion of the recombinant DNA encodes for a protein which, when expressed in a natural cellular host under the control of its natural promoter, is excreted in the medium of a culture of this natural cellular host.

The S gene of the hepatitis B virus genome constitutes a nucleotide sequence of particular interest in this regard, for several reasons. On the one hand, the expression product of the S gene in the cells which express it, HBsAg (11, 12), is secreted in the cell supernatant in the form of particles easy to detect and to quantify by radioimmunological assay, which allows a precise evaluation of the expression capacity of the viral vector. On the other hand, the invention provides a recombinant viral vector allowing the study of the expression of HBV genes both at the level of transcription and of translation, which is all the more interesting since To date, there has been no cell culture system capable of spreading the hepatitis B virus (HBV). Finally, cellular infection with the recombinant adenovirus-HBV virus illustrates in a particularly advantageous way the methodological basis of a process for manufacturing a vaccine against a determined pathogenic agent (in this case the hepatitis B virus in the 'example considered).

Another nucleotide sequence of the hepatitis B virus genome of particular interest is the S gene provided with its pre-S2 region which codes for the HBs antigen and for a receptor for polymerized human serum albumin (pHSA) (25 ), (26).

It goes without saying that the S gene can be substituted in the recombinant DNA by any other nucleotide sequence coding for a distinct protective antigen against another determined pathogenic agent, especially when this distinct protective antigen is itself normally capable of be secreted by cells transformed by recombinant DNA. It also goes without saying that the S gene and the pre-S2 region can be substituted in the recombinant DNA by any other nucleotide sequence coding for a distinct protective antigen against another determined pathogenic agent, especially when this distinct protective antigen is itself normally capable of being secreted by transformed cells by recombinant DNA. The nucleotide sequence coding for this distinct protective antigen can moreover possibly be inserted into the recombinant DNA in phase with another gene, for example the HBsAg antigen, since this other gene can be used as a "locomotive" to promote the excretion also of this distinct antigen, in particular in the form of a hybrid protein. By way of the separate antigens capable of being thus produced (where appropriate in the form of a hybrid protein), mention is made, for example, of glycoproteins of structure of the Epsteim-Barr virus.

The first nucleotides of the nucleotide sequence coding for a determined polypeptide (“simple” or hybrid protein) are placed, in particular by construction as close as possible to this promoter, in particular of the “TATA box”, characteristic of the promoter, it being understood however that the nucleotide sequence between the promoter and the ATG initiating the nucleotide sequence coding for said determined polypeptide must generally contain the triplets coding for the 5 'untranslated end of the messenger RNA normally corresponding to the coding sequence and containing the ribosome pairing sequences necessary for efficient translation. This 5 'untranslated end of the messenger RNA can moreover be replaced by the 5' untranslated end of a messenger RNA distinct from that normally associated with a determined coding sequence. For example, in the case of the S gene, it is possible to replace the 5 'untranslated end containing the pre-S gene or adjoining it with the 5' untranslated end of the messenger RNA of the T antigen from SV40. However, it has also been noted that when a DNA sequence containing the S and pre-S2 regions of the hepatitis B virus genome is used under the control of the strong E1A promoter, it is possible to obtain the expressions at the times from the pre-S2 region and from region S.
Any other 5 'untranslated end of messenger RNA can be used, as long as it is compatible with the other similar end chosen.

It is advantageous that the distance between the promoter's TATA box and the messenger RNA initiation site is approximately 30 nucleotides.

The promoter E1A of the recombinant DNA according to the invention and even more generally the vector according to the invention using larger parts of the genome of an adenovirus are preferably derived from an adenovirus belonging to category C, such that it has been defined by TOOZE. These adenoviruses have the known property of not being oncogenic. The Ad2 or Ad5 subtypes of this category of adenovirus are characterized by a significant transforming power. The use of the latter type of recombinant DNA is therefore particularly recommended, when the expression product sought is intended for the production of protective antigens, in particular active principles of vaccines. This will be all the more true in the case where whole, and even infectious, adenoviruses will be used as active principles of live vaccines, in particular under the conditions which will be explained further below.

The invention naturally also relates to cell lines, in particular of human or animal origin, which are transformed by recombinant DNAs as defined above and which have been made capable of synthesizing a polypeptide encoded by the nucleotide sequence (or said sequences nucleotides) contained in these recombinant DNAs and placed under the direct control of said promoter.

The invention relates more particularly still to cell lines transformed with a recombinant vector in accordance with the invention and further characterized in that the cells of these cell lines themselves contain a distinct sequence of nucleotides capable of complementing the part of the genome of the adenovirus which the above vector does not have, said distinct sequence preferably being incorporated into the genome of the cells of said cell line .

As such, the line 293 already mentioned above, after having been transformed by the recombinant vectors, constitutes a preferred cell culture according to the invention. Thanks to the complementation sequence which the cells of this line contain, a significant viral multiplication is observed inside these cells and, consequently, an equally multiplied expression of the coding sequence for the predetermined polypeptide. In the first case where this coding sequence is the S gene, a high production of HBsAg antigens is excreted in the culture medium of these cells. In the second case where the coding sequences consist of the S gene and the pre-S2 region of the hepatitis B virus, the recombinant adenovirus directs in vitro the synthesis of HBsAg particles having receptor activity for pHSA. When injected into rabbits, this recombinant virus produces anti-HBsAg and anti-pHSA antibodies. This shows the possibility of using a recombinant adenovirus to express a gene both in vitro and in vivo . The same vectors can be used for the transformation of Vero cells under analogous conditions.

The recombinant vectors according to the invention can also be used for the transformation of cells which do not itself have the complementation sequence under the conditions which have been indicated above. It may then be necessary to carry out a co-transformation of these latter types cells, on the one hand, with the recombinant vector according to the invention, on the other hand, with a non-defective adenovirus or a distinct recombinant DNA containing the adenovirus sequences which lack the recombinant vector according to the invention. It will certainly be observed in the latter case a simultaneous production of HBsAg antigens (when the coding sequence contains the S gene) and of the replicated adenovirus released by the cells thus transformed. The protective antigen formed may, however, if necessary, be separated from the viral suspension, for example by bringing the culture medium into contact with anti-adenovirus antibodies, preferably immobilized on a solid support, such as cross-linked agarose. , marketed under the designation SEPHAROSE. In any case, the presence of residual amounts of virus in the vaccine preparation is only of relatively minor importance. In fact, the adenovirus has only a weak pathogenic power in humans. It only causes mild respiratory infections.

The low importance of the pathogenic power of adenoviruses, more particularly those which belong to group C of human adenoviruses, makes it possible to envisage the constitution of "live vaccines". These can be constituted by infectious adenoviruses modified in the E3 region by the insertion of the recombinant DNA according to the invention into the nonessential part of the adenovirus. In this regard, it should be emphasized that human group C adenoviruses have never been shown to be tumorigenic in animals (3). These vectors or viruses will be of particular interest for the transformation of Vero cells, the non-tumorigenic character of which is now firmly established. Therefore they constitute a line of animal origin particularly favorable to the production of products for human use.

• la fig. 1 représente schématiquement les positions relatives des différentes régions du génome d'un adénovirus du sous-type Ad5, d'une part, et à échelle agrandie, de la région E1A de ce génome ;
• la fig. 2 est un schéma désormais classique du génome du virus de l'hépatite B ;
• les fig. 3 à 6 montrent les constructions successives qui ont conduit à la réalisation d'un plasmide contenant un premier ADN recombinant conforme à l'invention (fig. 6) et
• la fig. 7 représente schématiquement la construction d'un "virus recombinant défectif" à partir du plasmide modifié de la fig. 5 et de génomes défectifs de Ad5.

Additional characteristics of the invention will become apparent during the following description of preferred constructions of vectors containing the recombinant DNA according to the invention and of the conditions under which these vectors can be used. Reference will be made on this occasion to the drawings in which:

• fig. 1 schematically represents the relative positions of the different regions of the genome of an adenovirus of the Ad5 subtype, on the one hand, and on an enlarged scale, of the E1A region of this genome;
• fig. 2 is a now classic diagram of the genome of the hepatitis B virus;
• fig. 3 to 6 show the successive constructions which have led to the production of a plasmid containing a first recombinant DNA in accordance with the invention (FIG. 6) and
• fig. 7 schematically represents the construction of a "defective recombinant virus" from the modified plasmid of FIG. 5 and defective genomes of Ad5.

The following observations will first be made with regard to the figures, before describing the production of recombinant DNA constructions according to the invention.

In fig. 3 to 6, the parts in thin lines correspond to sequences of the plasmid pML2.

The numbers appearing in figs. 1 to 5 indicate the positions of the restriction sites in the viral sequences Ad5, SV40 and HBV. The numbering of the positions of the restriction sites of Ad5 and SV450 are those of J. TOOZE (1), those of HBV are those of P. TIOLLAIS et al. (11).

The erasures of the site designations in the drawings testify to the previous presence of the same sites in the corresponding parts of the DNAs which will be described below. These sites were however deleted, removed by repairing the cohesive ends of the fragments open or fragmented using the corresponding restriction enzymes, or by any other means as envisaged in the description which follows of the constructions which have been made.

1. Reminder of the main elements of the structure and organization of the Ad5 adenovirus genome (hereinafter often simply designated by the designation Ad5) :

• 1A : Le génome est une molécule de DNA linéaire double brin longue d'environ 36.000 pb. Les flèches indiquent la position et le sens des transcriptions des régions précoces E1a, E1b, E2, E3 et E4. Le promoteur majeur tardif PM_t et l'unité de transcription qui lui est associée sont également montrés. Les numérotations de 10 en 10, de 0 à 100, correspondent à des tailles exprimées en % de la taille du génome total.
• 1B : La région E1A. Les transcripts de cette région ont tous des extrémités 5'P (position 499) et 3'OH (position 1632) identiques. Le premier T de l'élément TATA du promoteur est situé à la position 468. Les sites de restriction utilisés dans les constrructions des plasmides sont indiqués. Les tailles sont exprimées par des nombres de paires de bases.

They result from parts 1A and 1B of FIG. 1.

• 1A : The genome is a double stranded linear DNA molecule approximately 36,000 bp long. The arrows indicate the position and the direction of the transcriptions of the early regions E1a, E1b, E2, E3 and E4. The late major promoter PM _t and the associated transcription unit are also shown. The numbers from 10 to 10, from 0 to 100, correspond to sizes expressed in% of the size of the total genome.
• 1B : The E1A region. The transcripts of this region all have identical 5'P (position 499) and 3'OH ends (position 1632). The first T of the TATA element of the promoter is located at position 468. The restriction sites used in the construction of the plasmids are indicated. Sizes are expressed by numbers of base pairs.

2. Origin of the fragments containing the S gene or the S gene and the pre-S2 region used in the constructions which follow . 1st case : Fragment of the hepatitis B virus genome containing the S gene.

It comes from the genome of the hepatitis B virus (fig. 2). It is recalled that the hepatitis B virus genome is a partially single-stranded circular DNA molecule. Its length is approximately 3,200 bp. It consists of the pairing of two strands of unequal length called strands L (-) and S (+). The S gene represents the coding sequence of the major polypeptide of the viral envelope which carries HBsAg. The DNA fragment used in the constructions below is the XhoI₁₂₇-BglII₁₉₈₄ fragment. The polyadenylation site of the HBsAg messenger was located at position 1916.

2nd case : Fragment of the genome of the hepatitis B virus containing the S gene and the pre-S2 region.

The DNA fragment used is the MstII₃₁₆₁-BglII₁₉₈₂ fragment which codes both for the HBs antigen and for a polymerized human serum albumin receptor (pHSA). The MstII site precedes the initiation codon of the pre-S2 region by 9 nucleotides. The BglII site is located 64 nucleotides downstream of the poly A addition signal of the S gene (from the English "poly A addition signal").

The construction and the propagation of a recombinant adenovirus comprising the S gene are discussed in the following. The procedure is identical for obtaining a recombinant adenovirus, in accordance with the invention, having the S gene and the pre- S2, it being understood that, in this second case, it is the DNA fragment MstII-BglII which is inserted between the HindIII and BamHI restriction sites of the plasmid pK4, in place of the DNA fragment XhoI-BglII of which it is question below.

The following will denote by Ad5 (X-B) the recombinant adenovirus having the S gene and by Ad5 (M-B) the recombinant adenovirus having the S gene and the pre-S2 region.

3. Construction of the plasmid pE1A (TagI) (fig. 3).

The plasmid pE1A (TaqI) contains the first 632 nucleotides of the left end of the Ad5 genome. This fragment was obtained by cutting the purified restriction fragment SacI E (0 - 5.0%) of Ad5 by TaqI (FIG. 1). This fragment was inserted between the EcoRI and ClaI restriction sites of the plasmid pML2 (fig. 3) Le plasmid pML2 was opened by EcoRI and Clal. The Ad5 fragment was linked to the linearized plasmid at the TaqI end. The junction of the TaqI-ClaI ends recreates a ClaI restriction site. The EcoRI end of the recombinant was repaired with DNA polymerase I from E. coli (Klenow fragment) and the plasmid recircularized using T4 ligase. The EcoRI site has therefore been reconstructed.

4. Manufacture of the plasmid pAB1 (FIG. 4).

The plasmid pAB1 was constructed from the plasmid pEIA (TaqI), so as to eliminate the coding part of the E1A region. This was done by isolating the PvuII-PvuII fragment (positions 452-623), cutting this fragment with the enzyme HaeIII (position 495), reinserting the PvuII₄₅₂-HaeIII₄₉₅ fragment at the PvuII₆₂₃ site of the plasmid pE1A (TaqI). In other words, the HaeIII₄₉₅-Pvu₆₂₃ fragment has been deleted. The plasmid pAB1 contains a HindIII site close to the transcription initiation site of the E1A region and a BamHI site (of pML2) located at a distance. These two restriction sites can be used to clone foreign genes without genetic fusion.

5. Production of the plasmid pK4 (FIG. 5).

pAB1 was cut with HindIII and BamHI and the fragment containing the E1A promoter, derived from pAB1, was linked, at its BamHI end to the BglI-BamHI fragment (positions 5235-2533 of the SV40 virus genome) hereinafter called A (SV40) containing the gene coding for the T and t antigens of the SV40 virus. After repair of the BglI and HindIII ends of the reconstitutor with the Kleenow fragment, the plasmid is recircularized using T4 ligase. The construction present in the plasmid pK4 was tested by bringing into play the transient expression of the T gene. Introduced into HeLa cells by transfection according to the calcium phosphate technique (19), the plasmid pK4 directs the synthesis of the antigen T of SV40 which was detected by immunofluorescence. About 1% of the transfected cells showed clear fluorescence. The absence of fluorescence after cell transfection with a plasmid containing the fragment of SV40 inserted in the wrong orientation shows that the gene for T and t antigens is well placed under the control of the E1A promoter of Ad5.

It is the HindIII site, at position 5171 of fragment A (SV40) which is then used to substitute the aforementioned fragment containing the gene 5 for most of A (SV40).

6. Production of the plasmid pK4S (XB) (fig. 6).

The plasmid pK4 was digested with HindIII and BamHI. The XhoI-BglII fragment (positions 125 to 1982) of the hepatitis B virus (HBV) genome (fig. 2) was inserted, in place of most of A (SV40) between the restriction sites HindIII and BamHI of the plasmid pK4, after repair of their respective ends by DNA polymerase I of E. coli (fragment of Kleenow) (fig. 3). The XhoI, HindIII, BamHI and BglII restriction sites are lost after ligation.

The HindIII site was located 8 nucleotides upstream of the ATG initiating the T and t antigens. The insertion of the S gene into this site therefore makes it possible to conserve the 5 end of the early SV40 mRNA containing the "capping" site and the messenger pairing sequences to the ribosomes. The HBV DNA fragment contains the coding sequence or S gene (position 155 to 833) of the major polypeptide of the viral envelope carrying HBsAg as well as the sequence located 3 ′ of the S gene and which includes the polyadenylation site of HBsAg messenger RNA at position 1916 (20, 21, 22). Two plasmids pK4S⁺ and pK4S⁻ carrying the HBV fragment inserted in both directions were isolated. 293 cells were transfected with these two plasmids and the synthesis of HBsAg was sought in the cell supernatant 3 days after the transfection. Only the plasmid pK4S⁺ which has the promoter E1A at the 5 'end of the S gene is capable of directing the synthesis of HBsAg. This shows that the expression of the S gene is well under the control of the E1A promoter of Ad5. Finally, a ClaI restriction site not methylated in E. coli was introduced into the plasmid pK4S⁺ (XB) at the NruI site of the sequence pML₂. The site is necessary for the construction of the recombinant virus.

It is finally the fragment delimited by PstI and ClaI ends, and obtained from pK4S⁺ (X-B), which was used for the manufacture of a "defective recombinant virus" in accordance with the invention.

7. Construction of the defective recombinant virus (fig. 7).

3 micrograms of the Pst1-ClaI restriction fragment purified from the plasmid pK4S⁺ (XB) were ligated to 20 micrograms of the ClaI restriction fragment (2.6% - 100%) of the Ad5 purified by sucrose gradient ultracentrifugation to provide the "recombinant defective virus" Ad5.

8. Pronation of the recombinant virus.

293 cells were grown in dishes 6 cm in diameter. 4 hours before transfection the culture supernatant was replaced with medium. 5 boxes of 293 cells at 70% confluence were then transfected with the ligation mixture according to the calcium phosphate technique then incubated for 4 hours at 37 ° C. After adsorption, the cells contained in each dish were washed with 2 ml of TS buffer (NaCl 8000.0 mg / l, KCl 380.0 mg / l, Na₂ HPO₄ 100.0 mg / l, CaCl₂ 100.0 mg / l, MgCl₂, 6H₂O 100.0 mg / l, Tris 3000.0 mg / l pH 7.4), treated with 400 microliters of a TS solution containing 20% glycerol for 1 minute at ordinary temperature, washed twice with 2 ml of TS buffer, then covered with 4 ml of MEM medium containing 1% noble agar, 1% fetal calf serum. On days 4 and 7, the cells were covered with 4 ml of the nutrient mixture. On day 10, the cells were stained with 4 ml of the nutrient medium supplemented with 0.01% neutral red. The plaques were observed on day 11. The viruses were resuspended in 1 ml of TS and amplified on 293 cells. The presence of HBsAG in the culture medium was tested by RIA (Austria II, laboratory ABBOTT). After amplification, the presence of HBV sequences in the recombinant was tested by hybridization. Five ranges were analyzed. Only one was against a recombinant HBsAg⁺ virus. This Ad5 clone (XB) as well as another clone were positive for the detection of HBV sequences. The size of the recombinant viral genome exceeds that of the wild virus by 2100 bp. No deletion could be detected by analysis of restriction fragments of the recombinant genome. Furthermore, this analysis showed that the pML2 sequences located between the PstI restriction site and the Ad5 sequence were correctly excised during the propagation of the recombinant genome in line 293.

9. The synthesis of HBsAg directed by the vector Ad5 (XB).

293 cells and Vero cells were infected with the Ad5 virus (XB). The expression levels of synthesized HBsAg are shown in Table I. Des samples of the cell supernatant were taken 3 days after infection and the HBsAg was sought by radioimmunoassay ("radioimmunoassay: RIA). The results show that the vector Ad5 (XB) is capable of directing the synthesis of HBsAg in these two cell lines, and the excretion of HBsAg by the cell lines in their respective culture media.

The synthesized HBsAg was purified by ultracentrifugation in CsCl. It has a density of 1.20. Typical particles of 22 nm have been observed by electron microscopy.

10. Synthesis of HBsAg directed by the Ad5 vectors (MB).

The expression levels of the HBsAg synthesized after infection of 293 cells and of Vero cells with Ad5 (M-B) are shown in Table I.

The kinetics of the extra- and intracellular distribution of HBsAg from Ver5 cells infected with Ad5 (M-B) indicated that the synthesis of HBsAg began 3 hours after infection and could be detected in the medium after 8 hours. Infection with the recombinant Ad5 virus (M-B) led to an accumulation of HBsAg of 0.5 to 1 µg / 10⁶ cells in the medium after 120 hours. Repeated experiments have shown that the recombinant virus containing the pre-S2 region synthesizes greater amounts of HBsAg than the recombinant virus containing only the S gene. HBsAg purified from the culture medium of cells infected with adenovirus recombinant Ad5 (MB) consisted of a homogeneous population of particles having an average diameter of 22 nm. The density after centrifugation in CsCl was 1.21.

11. Receptor activity for pHSA of HBsAq particles from Vero cells.

The HBsAg particles produced in Vero cells were tested by the hemagglutination technique of sheep red blood cells coated with pHSA and by radioimmunoassay (RIA) to detect the presence of pHSA receptor activity. Binding activity for pHSA was detected, but not for polymerized bovine albumin (Table II). Such activity was not detected with Vero cells infected with the recombinant Ad5 adenovirus (XB) containing only the S gene.

12. In vivo activity of the recombinant virus .

Rabbits were intravenously inoculated with highly purified preparations of the recombinant Ad5 adenovirus (M-B) and the wild type adenovirus. Although the HBsAg antigen could not be detected in their serum, 5 out of 8 rabbits, inoculated with the recombinant virus, showed the appearance of an anti-HBs titer varying from 20 to 270 mIU / ml after 15 days (Table III). No anti-HBs antibodies were detected in rabbits injected with the wild type of adenovirus. After a second intravenous inoculation carried out 4 weeks after the first, a second peak was observed in the anti-HBs response reaching 440 mIU / ml for one of the animals. 4 weeks after the second injection, anti-HBs titers varying from 6 mIU / ml to 360 mIU / ml were noted. Previous studies have indicated that the minimum level of anti-HBs still protective against HBV is 10 mIU / ml for humans. Anti-pHSA antibodies were tested in rabbits inoculated in order to determine their relationship with the neutralization of HBV. These antibodies, detected by the inhibition of hemagglutination, were found in 5 animals out of 5 having a positive anti-HBs response (Table IV).

The recombinant adenovirus Ad5 (MB) therefore directs in vivo the synthesis of HBsAg particles having a receptor character for polymerized human serum albumin.

The invention therefore provides a methodological basis for the manufacture of a hepatitis B vaccine (or against other types of conditions) in cell cultures.

The advantage of using adenovirus type 5 as a vector is twofold. On the one hand, this virus is a virus whose pathogenic power in humans is low. It only causes mild respiratory infections. On the other hand, this serotype which belongs to group C of human adenoviruses is not tumorigenic in animals. On the other hand, the rate of production of HBsAg obtained on Vero cells (approximately 1 microgram / 10⁶ / per infectious cycle) is a priori sufficient for industrial exploitation. However, this cell line is non-tumorigenic and for this reason, it constitutes the line of animal origin a priori the most favorable for the production of a product for human use.

It also goes without saying that 293 cells which have been mentioned above can be substituted for any other higher eukaryotic cells which can be infected with adenoviruses or which are capable of recognizing the E1A promoter of adenoviruses, these cells having been modified by prior incorporation into their own genomes, of a sequence containing the missing parts of the virus defective recombinant according to the invention of the genome of an adenovirus, in particular under the control of a promoter strongly recognized by these cells, for example a thymidine kinase promoter or an SV40 virus promoter. The sequence originating from the adenovirus, then integrated into the genome of these higher eukaryotic cells, can therefore complement the defective viruses in accordance with the invention, under conditions analogous to those permitted by 293 cells. These methods are applicable with a particular advantage to Vero cells.

The Ad5 adenovirus used was deposited on August 3, 1984 under the number I-322 at the C.N.C.M. ("National Collection of Cultures of Microorganisms from the INSTITUT PASTEUR of Paris.

Line 293 was deposited with the CN .CM on August 3, 1984 under the n ° I-323. TABLE I Production of extracellular HBsAg as a function of time Day Ad5 (XB) Infected Vero cells Ad5 (XB) 293 infected cells Ad5 (MB) Infected Vero cells Ad5 (MB) 293 infected cells 1 13 15 106 35 2 17 90 ND ND 3 45 110 349 56 4 110 100 668 60 5 320 130 1,057 63 6 470 120 1,153 ND 7 550 120 900 ND

TABLEAU III Réponse anti-HBs (mIU ml⁻¹) chez les lapins inoculés. Semaines Lapins 1 2 3 4 5 6 7 8 9 10 0 0 0 0 0 0 0 0 0 0 0 1 0 0 2 0 0 30 8 6 12 20 2 2 0 2 0 2 15 20 270 70 85 3 0 2 0 0 5 18 13 48 15 11 4 1 0 0 0 2 21 15 27 17 7 5 0 0 2 0 2 99 52 440 146 136 6 0 0 0 0 0 78 26 401 151 148 7 0 0 0 0 0 20 27 325 58 42 8 0 0 0 0 0 20 40 367 35 6
On a fait une injection intraveineuse de 10⁹ pfu de l'adénovirus Ad5 sauvage purifié à des lapins (lapins 1 et 2) et 10⁹ pfu d'adénovirus recombinant Ad5(M-B) purifié à des lapins (lapins 3 à 10) immédiatement après le prélèvement du sang à la semaine 0 et à la semaine 4. La quantité d'anticorps anti-HBs a été mesurée en utilisant le système RIA AUSAB de Abbott et exprimée en unités internationales (3,5 RIA équivalant à 1 mIU). (pfu = plage formant unité). TABLEAU IV Réponse immunogène du recepteur anti-pHSA (réciproque du titre). Semaines Lapins 6 7 8 9 10 0 0 0 0 0 0 1 16 2 2 2 2 2 4 16 16 16 16 3 4 4 16 8 8 4 2 2 4 4 4 5 16 32 32 32 32 6 16 32 32 16 32 7 8 8 32 8 16 8 4 4 8 4 8 The cumulative amounts of HBsAg (in ng) produced after infection of 293 cells and Vero cells, either by Ad5 (XB) or by Ad5 (MB) are indicated.

TABLE III Anti-HBs response (mIU ml⁻¹) in inoculated rabbits. Weeks Rabbits 1 2 3 4 5 6 7 8 9 10 0 0 0 0 0 0 0 0 0 0 0 1 0 0 2 0 0 30 8 6 12 20 2 2 0 2 0 2 15 20 270 70 85 3 0 2 0 0 5 18 13 48 15 11 4 1 0 0 0 2 21 15 27 17 7 5 0 0 2 0 2 99 52 440 146 136 6 0 0 0 0 0 78 26 401 151 148 7 0 0 0 0 0 20 27 325 58 42 8 0 0 0 0 0 20 40 367 35 6
An intravenous injection of 10⁹ pfu of the purified wild-type Ad5 adenovirus was given to rabbits (rabbits 1 and 2) and 10⁹ pfu of the purified recombinant ad5 adenovirus (MB) to rabbits (rabbits 3 to 10) immediately after collection. blood at week 0 and at week 4. The amount of anti-HBs antibodies was measured using the AUSAB RIA system from Abbott and expressed in international units (3.5 RIA equivalent to 1 mIU). (pfu = range forming unit). Immunogenic response of the anti-pHSA receptor (reciprocal of the titer). Weeks Rabbits 6 7 8 9 10 0 0 0 0 0 0 1 16 2 2 2 2 2 4 16 16 16 16 3 4 4 16 8 8 4 2 2 4 4 4 5 16 32 32 32 32 6 16 32 32 16 32 7 8 8 32 8 16 8 4 4 8 4 8

Rabbits were infected intravenously with 10⁹ pfu of purified Ad5 (M-B) recombinant adenovirus at weeks 0 and 4. The animals were identified by the same numbers as in Table III.

The anti-pHSA receptor activity has been expressed as the reciprocal of the highest serum titer capable of providing 100% inhibition of hemagglutination. The HBsAg particles having a receptor hemagglutination titer for the pHSA of 1: 128 were mixed with an equal volume of serial dilutions of inhibitory sera.

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Claims (23)

1. Recombinant vector containing the early promoter of the E1A region of the genome of an adenovirus for the transformation of eucaryotic, particularly human or animal cell lines, selected among those which are infectable by adenoviruses or whose endogenous polymerases are liable of recognizing the promoters of adenoviruses, said vector being further modified by a nucleic acid insertion containing a nucleotide sequence coding for a polypeptide sequence whose expression in said eucaryotic cell lines is sought, characterized in that said insertion sequence is substituted for the E1A genes normally placed under the direct control of said early promoter of the E1A region, said insertion sequence being itself placed under the direct control of said promoter, and in that it comprises, downstream of said nucleic acid insertion, all of the essential sequences of an adenovirus genome which are necessary for the replication of the corresponding adenovirus and which are normally located downstream of the genes normally under the direct control of the E1A early promoter in said genome, and in that said nucleotide sequence coding for the polypeptide sequence whose expression is sought is heterologous with respect to the genes of said adenovirus.
2. Recombinant vector according to claim 1, characterized in that the genome of the adenovirus with which it is associated is defective, in that it is deprived of the anterior part of the E1A region of the viral genome, particularly of its 0-2,6 % fragment.
3. Recombinant vector according to claim 1, characterized in that the recombinant DNA formed by said early promoter of the E1A region and the insertion nucleic sequence is incorporated in the non-essential region of an adenovirus, particularly in the non-essential part of the E3 region of said adenovirus.
4. Recombinant vector according to claim 1, characterized in that it consists of an infectious adenovirus comprising the recombinant DNA formed by the early promoter of the E1A region and the nucleic sequence insertion, incorporated in one of its non-essential regions, particularly in the non-essential part of the E3 region.
5. Recombinant vector according to any one of claims 1 to 4, characterized in that its promoter originates from an adenovirus of category C, belonging to sub-type Ad2 or Ad5.
6. Recombinant vector according to any one of claims 1 to 5, characterized in that the nucleotide sequence contained in the aforesaid nucleic acid insertion codes for a protein which, when it is expressed in a natural cellular host under the control of its natural promoter, is excreted into the culture medium of this natural cellular host.
7. Recombinant vector according to any one of claims 1 to 6, characterized in that the aforesaid nucleotide sequence codes for a protective antigen against a given pathogenic agent, particularly for the HBsAg antigen.
8. Recombinant vector according to claim 7, characterized in that said nucleic acid sequence comprises the S gene and the pre-S2 region of the genome of hepatitis B.
9. A cell line of human or animal origin, characterized in that it is transformed by a recombinant vector according to any one of claims 1 to 8, and in that it synthesizes the polypeptide coded by the aforesaid nucleotide sequence.
10. The cell line according to claim 9 considered in combination with claim 1 or claim 2 or both at once, and, as the case may be, with any one of claims 5 to 8, characterized in that it itself contains a distinct sequence of nucleotides able to complement the part of the genome of the adenovirus which the aforesaid vector is deprived of, the said distinct sequence preferably being incorporated into the genome of the cells of said cell line.
11. A process of production of a determined polypeptide including the transformation by a recombinant vector containing a nucleotide sequence coding for said polypeptide of the cells of a eucaryotic, particularly animal of human cell line, chosen among those which are infectable by the adenoviruses or whose endogenous polymerases are able to recognize the promoters of the adenoviruses, characterized by the fact that the transforming DNA is in accordance with any one of claims 1 to 8 and in that the expression products, including said specified polypeptide of these cells are recovered.
12. The process according to claim 11, characterized in that the recombinant vector is in accordance with any one of claims 5 to 8.
13. The process according to claim 11, characterized by the fact that the aforesaid transformed eucaryotic cells are in accordance with claim 10.
14. The process according to claim 11, characterized by the fact that the aforesaid transformed eucaryotic cells are simultaneously transformed with the corresponding infectious adenovirus.
15. The process according to any one of claims 11 to 14, characterized in that the specific nucleotide sequence is in accordance with that defined in claim 7.
16. A process of production of a polypeptide possessing the immunogenic properties characteristic of the virus of hepatitis B including the transformation of a line of eucaryotic, particularly animal or human cells, chosen from among those which are infectable by adenoviruses of whose endogenous polymerases are able to recognize the adenovirus promoters, by a recombinant vector containing an insert itself containing a nucleotide sequence coding for an immunogenic polypeptide characteristic of the virus of hepatitis B, characterized in that the aforesaid nucleotide sequence is substituted for the E1A genes normally placed under the direct control of the early promoter of the E1A region of the genome of an adenovirus, said insertion sequence being itself placed under the direct control of said promoter and said recombinant vector further containing, downstream of said nucleic acid insertion, all of the sequences of an adenovirus genome which are essential and necessary for the replication of the corresponding adenovirus and which are normally located downstream of the genes normally under the direct control of the early E1A promoter in said genome.
17. The process according to claim 16, characterized in that the recombinant vector consists of a defective andenovirus which can be replicated in said cell lines, said adenovirus being deprived of the anterior part of the E1A region of the viral genome, particularly of its 0-2,6 % fragment, and in that one recovers in the culture medium the expression products of the eucaryotic cell lines transformed by the recombinant DNA, including the polypeptide coded by the aforesaid nucleotide sequence and synthesized by said cell lines.
18. The process according to claims 16 to 17, characterized in that the promoter of the recombinant DNA originates from an adenovirus of category C, belonging to sub-type Ad2 or Ad5.
19. The process according to any one of claims 16 to 18, characterized in that the recombinant DNA formed by said early promoter and said insertion sequence is incorporated into a non-essential region of the adenovirus, particularly into the non-essential part of the E3 region of this adenovirus.
20. The process according to any one of claims 16 to 19, characterized in that the eucaryotic cell line itself contains a distinct sequence of nucleotides able to complement the part of the genome of the adenovirus of which the aforesaid vector is deprived, the said sequence preferably being incorporated into the genome of the cells of said cell line.
21. The process according to any one of claims 16 to 20, characterized in that the recombinant DNA includes a nucleotide sequence corresponding to the S gene of the genome of the virus of hepatitis B.
22. The process according to any one of claims 16 to 20, characterized in that the recombinant DNA includes the sequence corresponding to the S gene and the pre-S2 region of the genome of the virus of hepatitis B.
23. Vaccine whose active principle consists of an adenovirus which is infectious and can be replicated and which comprises, incorporated into one of its non-essential regions, particularly the non-essential part of its E3 region, a DNA recombinant formed between the early promoter of the E1A region and a nucleic sequence insertion coding for a protective antigen against the pathogenic agent selected, and particularly against the hepatitis B virus, said nucleic sequence insertion being substituted for the E1A genes normally under the control of said promoter.

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